Fluorescent dyes or tags are used in various applications as highly sensitive detection reagents. Fluorescent dyes or tags that preferentially label a selected component of a sample can be used to determine the presence, quantity or location of that selected component. For example, thiol reactive probes such as BODIPY, fluorescein, Oregon Green, tetramethylrhodamine and Texas Red are use routinely to fluorescently label proteins and polypeptides as these probes react readily with the thiol groups, also called mercaptans or sulfhydryls, present in cysteine residues. Additional examples of thiol reactive probes are set forth in Chapter 2 of the commercial catalog for Molecular Probes, Inc. (Richard Haugland, Handbook of Fluorescent Probes and Research Chemicals, 6th Edition 1996 Molecular Probes, Eugene, Oreg., pp. 47–62).
Various other compounds for use as fluorescent tags have also been described. See, for example, U.S. Pat. No. 6,335,446 describing quinolinium- and pyridinium-based fluorescent dyes; U.S. Pat. No. 6,299,055 describing fluorinated xanthine derivatives; U.S. Pat. No. 6,291,203, U.S. Pat. No. 5,658,735 and U.S. Pat. No. 5,268,486 describing cyanine dyes; U.S. Pat. No. 5,654,442 and U.S. Pat. No. 5,188,934 describing 4,7-dichlorofluorescein dyes; U.S. Pat. No. 5,451,663, U.S. Pat. No. 5,248,782 and U.S. Pat. No. 5,274,113 describing heteroaryl-substituted dipyrrometheneboron difluoride dyes and U.S. Pat. No. 5,433,896 describing dibenzopyrrometheneboron difluoride dyes; U.S. Pat. No. 5,583,236 and U.S. Pat. No. 5,371,241 describing fluorescein labeled phosphoramidites; U.S. Pat. No. 5,437,980 describing phenanthridium dyes; and U.S. Pat. No. 5,442,045 describing biological conjugates of fluorescent rhodol dyes.
Coumarin derivatives are used widely as fluorescent labels or tracers and in the preparation of fluorogenic substrates for enzymes, particularly when a fluorophore having excitation in the ultraviolet and generally blue to blue-green fluorescent emission range is desired. U.S. Pat. No. 4,956,480 discloses 3-carboxyalkyl derivatives of 7-amino-4-methylcoumarin and U.S. Pat. No. 4,279,992 discloses 7-hydroxycoumarin or umbelliferone for use as fluorescent labels or tags. However coumarin dyes are not particularly soluble in the aqueous environment of most biological systems. Further, their fluorescence is often unstable over periods of extended use and is quenched upon conjugation, particularly to proteins.
U.S. Pat. No. 5,696,157 discloses fluorescent sulfonated derivatives of 7-aminocoumarin that remain bright upon conjugation and exhibit photostability and enhanced aqueous solubility.
Furocoumarin compounds, more commonly known as psoralens, have been used for centuries in combination with ultraviolet light in cosmetics and for the treatment of proliferative skin disorders such as eczema, vitiligo, psoriasis, and mycosis fungoides. More recently, this photosensitive therapy has been used to treat cancer, in particular T cell lymphoma, autoimmune diseases, and microbial infection.
Psoralens intercalate into DNA in the cell nucleus and subsequently enter into photo-induced crosslinking with the DNA by forming 2+2, cyclobutane fusions from double-bonds in the psoralen to double bonds in the pyrimidine bases.
Psoralens (furocoumarins) can also be used as photo reactive nucleic acid stains for probing DNA and RNA structures. 4,5′,8-Trimethylpsoralen, psoralen, 5-methoxypsoralen and 8-methoxypsoralen intercalate into double-stranded DNA where, upon illumination with ultraviolet light of wavelengths from 320 to 400 nm (ultraviolet light A or UVA), they covalently bind to pyrimidines (Annals NY Acad. Sci. 1980 346:355). However, like other coumarins, the intrinsic blue fluorescence of these compounds is usually quenched upon binding to the target.
5′-substituted, 4′,5′-dihydropsoralen compounds bearing organomercurial moieties and methods for their production are described in U.S. Pat. No. 6,255,324, the teachings of which are herein incorporated by reference in its entirety. These compounds are taught to be useful in the treatment of proliferative skin disorders and diseases of the blood and bone marrow, as well as antimicrobial agents.
It has now been found that these mercurimethyl-dihydropsoralen compounds and derivatives thereof are highly reactive with thiol containing species. Further, the reaction product is highly fluorescent. Accordingly, these compounds are useful as fluorescent tags for labeling and detection of thiol containing species, in particular amino acids, proteins and peptides. In addition, the fluorescent thiol containing reaction products maintain their ability to intercalate into nucleic acid sequences, thus also providing a fluorescent tag for labeling and detection of nucleic acids.